Regulatory sequences for modulating transgene expression in plants

ABSTRACT

The invention relates to gene expression regulatory sequences, specifically introns that act as enhancers of gene expression, the promoter and terminator sequences endogenously associated with these introns. Presence of these intronic enhancer sequences in proximity to promoter sequences leads to enhancement of gene expression. Methods of finding such new intronic enhancer sequences and using them to generate transgenic plants are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Indian Provisional ApplicationNo. 1340/DELNP/2010, filed Jun. 9, 2010, and U.S. ProvisionalApplication No. 61/372,515, filed Aug. 11, 2010, the entire contents ofeach is herein incorporated by reference.

FIELD OF INVENTION

The present invention relates to the generation of transgenic plants,particularly to the use of promoter and intron sequences to regulategene expression in plants.

BACKGROUND

Recent advances in plant genetic engineering have opened new doors toengineer plants to have improved characteristics or traits. Thesetransgenic plants characteristically have recombinant DNA constructs intheir genome that have a protein-coding region operably linked to atleast one regulatory region that is the promoter. The promoter can be astrong or weak promoter, or a constitutive or tissue-specific promoter.Besides the promoter, the expression level of the gene product can bemodulated by other regulatory elements such as introns. Introns areintervening, non-coding sequences that are present in most eukaryoticgenes. Introns have been reported to affect the levels of geneexpression. This effect is known as Intron Mediated Enhancement (IME) ofgene expression (Lu at al., Mol Genet Genomics (2008) 279:563-572).Callis et al., (Genes Dev. 1987 1:1183-1200) showed that the presence ofthe first intron from maize alcohol dehydrogenase-1 (Adh1) geneincreased the expression levels of transgenes in cultured maize cells upto 100-fold when compared to intronless constructs. Mascarenkas et al.(Plant Mol. Biol., 1990, 15: 913-920) showed that other introns from themaize Adh1 gene could also increase heterologous gene expression inmaize protoplasts. Vasil at al. (Plant Physiol., 1989, 91:1575-15790)reported that the constructs containing Shrunken-1 (Sh-1) first intronhad much higher expression levels of the reporter gene in plantprotoplasts, when compared to the constructs with promoter alone, or toconstructs with promoter and Adh-1 first intron. Identifying novelregulatory sequences can lead to finer modulation of gene expression intransgenic plants.

Plant genetic engineering has advanced to introducing multiple traitsinto commercially important plants, also known as gene stacking. This isaccomplished by multigene transformation, where multiple genes aretransferred to create a transgenic plant that might express a complexphenotype, or multiple phenotypes. But it is important to modulate orcontrol the expression of each transgene optimally. The regulatoryelements such as the promoter and the terminator sequences need to bediverse, to avoid introducing into the same transgenic plant repetitivesequences, which has been correlated with undesirable negative effectson transgene expression and stability (Peremarti et al (2010) Plant MolBiol 73:363-378; Mette et al (1999) EMBO J 18:241-248; Matte at al(2000) EMBO J. 19:5194-5201; Mourrain et al (2007) Planta 225:365-379,U.S. Pat. No. 7,632,982, U.S. Pat. No. 7,491,813, U.S. Pat. No.7,674,950, PCT Application No. PCT/US2009/046968). Therefore it isimportant to discover and characterize novel regulatory elements thatcan be used to express heterologous nucleic acids in important cropspecies. Diverse regulatory regions can be used to control theexpression of each transgene optimally.

SUMMARY

The present invention relates to regulatory sequences for modulatinggene expression in plants. Recombinant DNA constructs comprisingregulatory sequences are provided. Recombinant DNA constructs comprisingintron sequences acting as enhancers of gene expression and endogenouspromoter and terminator sequences corresponding to these intronsequences are provided.

Another embodiment of the invention is a recombinant DNA constructcomprising an intron operably linked to a promoter and a terminatorwherein the intron comprises a nucleotide sequence that has at least 95%sequence identity to SEQ ID NO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101,102, 103, 104, 118, 137 or 138. In another embodiment, the introncomprises the nucleotide sequence of SEQ ID NO: 4, 8, 13, 19, 52, 53,56, 57, 58, 101, 102, 103, 104, 118, 137 or 138.

One embodiment of the invention is a recombinant DNA constructcomprising an intron operably linked to a promoter and a terminatorwherein the promoter comprises a nucleotide sequence that has at least95% sequence identity to SEQ ID NO: 105-117, 119, 136 or 139. In anotherembodiment, the promoter comprises the nucleotide sequence of SEQ ID NO:105-117, 119, 136 or 139.

One embodiment of the invention is a recombinant DNA constructcomprising intron operably linked to a promoter and a terminator whereinthe terminator comprises a nucleotide sequence that has at least 95%sequence identity to SEQ ID NOS: 140, 141, 142 or 143. In anotherembodiment, the terminator comprises the nucleotide sequence of SEQ IDNO: 140, 141, 142 or 143.

One embodiment of the invention is a recombinant DNA constructcomprising an intron operably linked to a promoter and a terminatorwherein the intron comprises a nucleotide sequence that has at least 95%identity to SEQ ID NOS: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103,104, 118, 137 or 138; and the promoter comprises a nucleotide sequencethat has at least 95% identity to SEQ ID NOS: 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 119, 136 or 139.

One embodiment of the invention is a recombinant DNA constructcomprising an intron operably linked to a promoter and a terminatorwherein the intron comprises a nucleotide sequence that has at least 95%identity to SEQ ID NO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103,104, 118, 137 or 138; the promoter sequence has at least 95% identity toSEQ ID NO: 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 119, 136 or 139; and the terminator has at least 95% sequenceidentity to SEQ ID NO: 140, 141, 142 or 143.

In one embodiment of the current invention, the intron is operablylinked to the promoter, and is present downstream of the promoter, inthe recombinant DNA constructs described herein. One embodiment of thepresent invention includes a recombinant DNA construct comprising anintron described in the present invention, operably linked to a promoterand a heterologous polynucleotide, wherein the intron can act asenhancer of expression of the heterologous polynucleotide.

Another embodiment of the invention encompasses a recombinant DNAconstruct comprising an intron wherein the intron sequence comprises atleast one copy of the 8-bp sequence motif of SEQ ID NO: 99; or containsat least one copy of the 8-bp sequence motif of SEQ ID NO: 99 and atleast one copy of the 5-bp sequence motif of SEQ ID NO: 100, wherein theintron is capable of enhancing expression of a heterologouspolynucleotide in a transgenic plant. The intron sequence can alsocomprise more than one copy of SEQ ID NO: 99, or can comprise one ormore than one copy of SEQ ID NO: 99 and more than one copy of SEQ ID NO:100.

Another embodiment of this invention is a method to identify novelintrons that are useful for enhancing expression of a heterologouspolynucleotide in a plant cell, the method comprising the steps ofscanning a plurality of introns from plants for presence of SEQ ID NO:99, selecting a sequence that contains at least one copy of SEQ ID NO:99, measuring the efficacy of the identified intron to enhanceexpression of a heterologous polynucleotide in a plant.

Another embodiment of the invention is a method for identifying novelintronic sequences for enhancing transgene expression inmonocotyledenous plants by identifying sequences orthologous to SEQ IDNO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103, 104, 118, 137 or138; and measuring the enhancing effect of the identified intron on theexpression of an operably linked heterologous polynucleotide.

Another embodiment of the current invention includes the promoter andthe terminator sequences that are endogenously linked to the intronsidentified using the methods described in the current invention.

Another embodiment of the current invention is a method for modulatingexpression of a heterologous polynucleotide in a monocotyledonous plantcomprising the steps of: (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a promoter and a heterologouspolynucleotide wherein each is operably linked to an intron, wherein theintron comprises either (i) a nucleotide sequence that is orthologous toSEQ ID NO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103, 104, 118,137 or 138; or (ii) a nucleotide sequence that contains least one copyof a sequence motif identical to SEQ ID NO: 99; and (b) regenerating atransgenic plant from a regenerable monocotyledonous plant cell afterstep (a) wherein the transgenic plant comprises the recombinant DNAconstruct; and (c) obtaining a progeny plant derived from the transgenicplant of step (b), wherein said progeny plant comprises the recombinantDNA construct and exhibits enhanced expression of the heterologouspolynucleotide when compared to a plant comprising a correspondingrecombinant DNA construct without the intron sequence.

In another embodiment, this invention concerns a vector, cell, plant, orseed comprising a recombinant DNA construct comprising the regulatorysequences described in the present invention.

The invention encompasses regenerated, mature and fertile transgenicplants comprising the recombinant DNA constructs described above,transgenic seeds produced therefrom, T1 and subsequent generations. Thetransgenic plant cells, tissues, plants, and seeds may comprise at leastone recombinant DNA construct of interest.

In one embodiment, the plant comprising the regulatory sequencesdescribed in the present invention is a monocotyledenous plant. Inanother embodiment, the plant comprising the regulatory sequencesdescribed in the present invention is a maize plant.

BRIEF DESCRIPTION OF DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application. The Sequence Listing contains the oneletter code for nucleotide sequence characters and the three lettercodes for amino acids as defined in conformity with the IUPAC-IUBMBstandards described in Nucleic Acids Research 13:3021-3030 (1985) and inthe Biochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. §1.822.

FIG. 1 is a schematic representation of the vector used for testingintrons showing the location of restriction sites used to clone intronsrelative to the maize ubiquitin promoter, as described in Example 2.

FIG. 2 shows the map of PHP 41353, the ITVUR-2 vector used for testingintron-mediated enhancement of gene expression.

FIG. 3 shows quantitative analysis of GUS reporter gene expression inMaize Embryos infected with the respective constructs.

FIG. 4 shows the fold enhancement of GUS reporter gene expression inrice calli infected with intron constructs when compared with thecontrol vector ITVUR-2.

FIG. 5 shows the map of PHP38808, the vector with CYMV promoter and ADH1intron, used for testing intron-mediated enhancement of gene expression,as described in Example 7.

FIG. 6 shows the results of Northern blot of RNA extracted frominfiltrated maize tissue culture material and probed with adigoxigenin-labeled DNA probe for the insecticidal gene used. Sampleswere loaded based on ELISA data to contain equal amounts of PAT.

FIG. 7 shows the map of PHP34651, vector containing GATEWAY® attRrecombination sites and a PAT expression cassette used for LR reactionsto generate the final expression vectors for introns, as described inExample 7.

FIG. 8 shows the map of PHP42365, vector containing ZmUbi promoter andZmUbi intron, for testing in stable transgenic rice plants, as describedin Example 11.

FIG. 9 shows MUG data from stable transgenic lines transformed withdifferent constructs. Data represents the average of 5-8 independentsingle copy events±SE.

FIG. 10 shows MUG data from stable transgenic lines transformed withdifferent constructs. Data represents the average of 5-8 independentsingle copy events±SE.

FIG. 11 shows histochemical data from leaves and panicles collected fromstable transgenic lines transformed with different constructs.Representative images are shown for each construct analyzed.

FIG. 12 shows histochemical data from leaves and panicles collected fromstable transgenic lines transformed with different constructs.Representative images are shown for each construct analyzed.

FIG. 13 is the schematic representation of the PHP49597 vector(terminator test vector).

SEQ ID NO: 1 is the sequence of the maize ubiquitin promoter.

SEQ ID NO: 2 is the sequence of the first intron from maize ubiquitingene.

SEQ ID NO: 3 is the nucleotide sequence of PHP41353, ITVUR-2 vector.

SEQ ID NOS: 4-19 and SEQ ID NOS: 52-58, SEQ ID NO: 118, SEQ ID NOS: 137and 138 are sequences of introns that were tested to identifyexpression-enhancing introns, and are described in Table 1 below.

SEQ ID NOS: 105-113, SEQ ID NO: 119 and SEQ ID NOS: 136 and 139 are thesequences of promoters identified for the enhancing introns as describedin Example 10 and Example 11, and are described in Table 1 below.

SEQ ID NOS: 140-143 given in Table 1 are the sequences of the endogenousterminators for the introns TS1, TS2, TS13 and TS27, identified asexplained in Example 13.

TABLE 1 SEQ ID Intron/ Enhancing/Non-Enhancing NO Name Promoter Intron 4TS1 Intron Enhancing 5 TS4 Intron Non-Enhancing 6 TS5 IntronNon-Enhancing 7 TS6 Intron Non-Enhancing 8 TS7 Intron Enhancing* 9 TS8Intron Non-Enhancing 10 TS10 Intron Non-Enhancing 11 TS11 IntronNon-Enhancing 12 TS12 Intron Non-Enhancing 13 TS13 Intron Enhancing 14TS14 Intron Non-Enhancing 15 TS15 Intron Non-Enhancing 16 TS16 IntronNon-Enhancing 17 TS17 Intron Non-Enhancing 18 TS24 Intron Non-Enhancing19 TS27 Intron Enhancing* 52 i1 Intron Enhancing 53 i2 Intron Enhancing54 i3 Intron Non-Enhancing 55 i4 Intron Non-Enhancing 56 i5 IntronEnhancing 57 i6 Intron Enhancing 58 i7 Intron Enhancing 105 pTS1Promoter Promoter identified for SEQ ID NO: 4 106 pTS7 Promoter Promoteridentified for SEQ ID NO: 8 107 pTS13 Promoter Promoter identified forSEQ ID NO: 13 108 pTS27 Promoter Promoter identified for SEQ ID NO: 19109 pi1 Promoter Promoter identified for SEQ ID NO: 52 110 pi2 PromoterPromoter identified for SEQ ID NO: 53 111 pi5 Promoter Promoteridentified for SEQ ID NO: 56 112 pi6 Promoter Promoter identified forSEQ ID NO: 57 113 pi7 Promoter Promoter identified for SEQ ID NO: 58 118TS2 Intron Enhancing 119 pTS2 Promoter Promoter identified for SEQ IDNO: 118 136 pTS1v Promoter Promoter sequence cloned for SEQ ID NO: 4 137TS7v Intron Enhancing 138 TS27v Intron Enhancing 139 pTS27v PromoterPromoter sequence cloned for SEQ ID NO: 19 140 tTS1 TerminatorTerminator identified for SEQ ID NO: 4 141 tTS2 Terminator Terminatoridentified for SEQ ID NO: 118 142 tTS13 Terminator Terminator identifiedfor SEQ ID NO: 13 143 tTS27 Terminator Terminator identified for SEQ IDNO: 19 *based on results from variants

SEQ ID NOS: 20-51 are the primers used for cloning introns as describedin Table 2 in Example 3.

SEQ ID NO: 59 is the sequence of the vector PHP38808, used for testingintron-mediated enhancement of gene expression as described in Example7. SEQ ID NO: 60 is the sequence of PHP34651, the vector containingGATEWAY® attR recombination sites and a PAT expression cassette used forLR reactions to generate the final expression vectors for introns, asdescribed in Example 7.

SEQ ID NOS: 61-94 are the oligonucleotides used for generating intronsby oligonucleotide stacking as described in Table 4 in Example 7.

SEQ ID NO: 95 is the sequence for first intron of adh1 gene.

SEQ ID NO: 96 is the sequence for intron 6 for adh1 gene.

SEQ ID NO: 97 is the sequence for intron 1 for shrunken1 (Sh-1) gene

SEQ ID NO: 98 is the sequence for ubi intron 1 used for computationalanalyses as described in Example 8.

SEQ ID NO: 99 is the sequence of the 8-bp motif identified as describedin Example 8.

SEQ ID NO: 100 is the sequence of the 5-bp motif identified as describedin Example 8.

SEQ ID NOS: 101-104 are the intron sequences containing the 8-bp motif(SEQ ID NO: 99), as described in Example 9.

SEQ ID NOS: 114-117 are the promoter sequences identified from theintrons of SEQ ID NOS: 101-104 respectively, as described in Examples 9and 10.

SEQ ID NOS: 120-128 are the sequences of the primers used for cloningthe promoters and introns, as described in Table 7.

SEQ ID NOS: 129-134 are the primer and probe sequences for qPCR, asdescribed in Table 9 and Table 10.

SEQ ID NO: 135 is the sequence of the PHP42365 vector that containsZmUbi promoter and ZmUbi intron.

SEQ ID NO: 144 is the sequence of the PHP49597 vector (terminator testvector or TTV).

SEQ ID NO: 145 corresponds to the nucleotide sequence GATCAAAAAAAAAAAAAof a ‘promiscuous’ MPSS tags.

SEQ ID NO: 146 corresponds to the nucleotide sequence of a consensusmotif sequence, which encompasses variations of the motif sequence givenin SEQ ID NO: 99.

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUBMB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended cairns, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein:

The terms “monocot” and “monocotyledonous plant” are usedinterchangeably herein. A rnonocot of the current invention includes theGramineae.

The terms “dicot” and “dicotyledonous plant” are used interchangeablyherein. A dicot of the current invention includes the followingfamilies: Brassicaceae, Leguminosae, and Solanaceae.

The terms “full complement” and “full-length complement” are usedinterchangeably herein, and refer to a complement of a given nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary.

“Transgenic” refers to any cell, cell line, callus, tissue, plant partor plant, the genome of which has been altered by the presence of aheterologous nucleic acid, such as a recombinant DNA construct,including those initial transgenic events as well as those created bysexual crosses or asexual propagation from the initial transgenic event.The term “transgenic” as used herein does not encompass the alterationof the genome (chromosomal or extra-chromosomal) by conventional plantbreeding methods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. For example, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably to refer to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA that is without introns andthat can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from anmRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase I.

An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from acDNA library and therefore is a sequence which has been transcribed. AnEST is typically obtained by a single sequencing pass of a cDNA insert.The sequence of an entire cDNA insert is termed the “Full-InsertSequence” (“FIS”). A “Contig” sequence is a sequence assembled from twoor more sequences that can be selected from, but not limited to, thegroup consisting of an EST, FIS and PCR sequence. A sequence encoding anentire or functional protein is termed a “Complete Gene Sequence”(“CGS”) and can be derived from an FIS or a contig.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or pro-peptides present in the primarytranslation product has been removed.

“Precursor” protein refers to the primary product of translation ofmRNA; i.e., with pre- and pro-peptides still present. Pre- andpro-peptides may be and are not limited to intracellular localizationsignals.

“Isolated” refers to materials, such as nucleic acid molecules and/orproteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleolides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acidfragments that are not normally found together in nature. Accordingly, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that normally found in nature.

The terms “entry clone” and “entry vector” are used interchangeablyherein.

The term “insecticidal gene” and “insect resistance gene” are usedinterchangeably herein.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

“Expression” refers to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in a nullsegregating (or non-transgenic) organism from the same experiment.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct) into a cell, means “transfection” or“transformation” or “transduction” and includes reference to theincorporation of a nucleic acid fragment into a eukaryotic orprokaryotic cell where the nucleic acid fragment may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation andtransient transformation.

“Stable transformation” refers to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentis stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

The term “crossed” or “cross” means the fusion of gametes viapollination to produce progeny (e.g., cells, seeds or plants). The termencompasses both sexual crosses (the pollination of one plant byanother) and selfing (self-pollination, e.g., when the pollen and ovuleare from the same plant). The term “crossing” refers to the act offusing gametes via pollination to produce progeny.

A “favorable allele” is the allele at a particular locus that confers,or contributes to, a desirable phenotype, e.g., increased cell walldigestibility, or alternatively, is an allele that allows theidentification of plants with decreased cell wall digestibility that canbe removed from a breeding program or planting (“counterselection”). Afavorable allele of a marker is a marker allele that segregates with thefavorable phenotype, or alternatively, segregates with the unfavorableplant phenotype, therefore providing the benefit of identifying plants.

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct/expression construct) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the MEGALIGN® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the Clustal V method of alignment(Higgins and Sharp, CABIOS. 5:151-153 (1989)) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments and calculation of percent identity of proteinsequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters areKTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignmentof the sequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

The present invention includes a polynucleotide comprising: (i) anucleic acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity, based on the Clustal V methodof alignment, when compared to SEQ ID NOS: 4, 8, 13, 19, 52, 53, 56, 57,58, 101-119, 136-143; or (ii) a full complement of the nucleic acidsequence of (i), wherein the polynucleotide acts as a regulator of geneexpression in a plant cell.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

Regulatory Sequences:

A recombinant DNA construct (including a suppression DNA construct) ofthe present invention may comprise at least one regulatory sequence.

“Regulatory sequences” or “regulatory elements” are used interchangeablyand refer to nucleotide sequences located upstream (5″ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include, but are not limited to, promoters, translationleader sequences, introns, and polyadenylation recognition sequences.The terms “regulatory sequence” and “regulatory element” are usedinterchangeably herein.

“Promoter” refers to a nucleic acid fragment capable of controllingtranscription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably to refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” refers to a promoter whose activityis determined by developmental events.

Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”.

High level, constitutive expression of the candidate gene under controlof the 35S or UBI promoter may have pleiotropic effects, althoughcandidate gene efficacy may be estimated when driven by a constitutivepromoter. Use of tissue-specific and/or stress-specific promoters mayeliminate undesirable effects but retain the ability to enhance droughttolerance. This effect has been observed in Arabidopsis (Kasuga et al.(1999) Nature Biotechnol. 17:287-91).

Suitable constitutive promoters for use in a plant host cell include,but are not limited to, the core promoter of the Rsyn7 promoter andother constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 355 promoter (Odell et al., Nature 313:810-812(1985)); rice actin (McElroy at al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen at al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last atal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten at al., EMBO J.3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, but are not limited to, forexample, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and6,177,611.

In choosing a promoter to use in the methods of the invention, it may bedesirable to use a tissue-specific or developmentally regulatedpromoter.

A tissue-specific or developmentally regulated promoter is a DNAsequence which regulates the expression of a DNA sequence selectively inthe cells/tissues of a plant critical to tassel development, seed set,or both, and limits the expression of such a DNA sequence to the periodof tassel development or seed maturation in the plant. Any identifiablepromoter may be used in the methods of the present invention whichcauses the desired temporal and spatial expression.

Promoters which are seed or embryo-specific and may be useful in theinvention include, but are not limited to, soybean Kunitz trypsininhibitor (Kti3, Jofuku and Goldberg, Plant Cell 1:1079-1093 (1989)),patatin (potato tubers) (Rocha-Sosa, M., at al. (1989) EMBO J. 8:23-29),convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W. G., et al.(1991) Mol. Gen. Genet. 259:149-157; Newbigin, E. J., et al. (1990)Planta 180:461-470; Higgins, T. J. V., et al. (1988) Plant. Mol. Biol.11:683-695), zein (maize endosperm) (Schemthaner, J. P., et al. (1988)EMBO J. 7:1249-1255), phaseolin (bean cotyledon) (Segupta-Gopalan, C.,et al. (1985) Proc. Natl. Acad. Sci, U.S.A. 82:3320-3324),phytohemagglutinin (bean cotyledon) (Voelker, T. et al. (1987) EMBO J.6:3571-3577), B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L,et al. (1988) EMBO J. 7:297-302), glutelin (rice endosperm), hordein(barley endosperm) (Morris, C., et al. (1988) Plant Mol. Biol.10:359-366), glutenin and gliadin (wheat endosperm) (Colot, V., et al.(1987) EMBO J. 6:3559-3564), and sporamin (sweet potato tuberous root)(Hattori, T., et al. (1990) Plant Mol. Biol. 14:595-604). Promoters ofseed-specific genes operably linked to heterologous coding regions inchimeric gene constructions maintain their temporal and spatialexpression pattern in transgenic plants. Such examples include, but arenot limited to, Arabidopsis thaliana 2S seed storage protein genepromoter to express enkephalin peptides in Arabidopsis and Brassicanapus seeds (Vanderkerckhove et al., Bio/Technology 7:L929-932 (1989)),bean lectin and bean beta-phaseolin promoters to express luciferase(Riggs et al., Plant Sci. 63:47-57 (1989)), and wheat glutenin promotersto express chloramphenicol acetyl transferase (Colot et al., EMBO J.6:3559-3564 (1987)).

Inducible promoters selectively express an operably linked DNA sequencein response to the presence of an endogenous or exogenous stimulus, forexample by chemical compounds (chemical inducers) or in response toenvironmental, hormonal, chemical, and/or developmental signals.Inducible or regulated promoters include, but are not limited to, forexample, promoters regulated by light, heat, stress, flooding ordrought, phytohormones, wounding, or chemicals such as ethanol,jasmonate, salicylic acid, or safeners.

For instance, introns of the present invention can be combined withinducible promoters to enhance their activity without affecting theirinducibility characteristics.

A minimal or basal promoter is a polynucleotide molecule that is capableof recruiting and binding the basal transcription machinery. One exampleof basal transcription machinery in eukaryotic cells is the RNApolymerase II complex and its accessory proteins.

Plant RNA polymerase II promoters, like those of other highereukaryotes, are comprised of several distinct “cis-actingtranscriptional regulatory elements,” or simply “cis-elements,” each ofwhich appears to confer a different aspect of the overall control ofgene expression. Examples of such cis-acting elements include, but arenot limited to, such as TATA box and CCAAT or AGGA box. The promoter canroughly be divided in two parts: a proximal part, referred to as thecore, and a distal part. The proximal part is believed to be responsiblefor correctly assembling the RNA polymerase II complex at the rightposition and for directing a basal level of transcription, and is alsoreferred to as “minimal promoter” or “basal promoter”. The distal partof the promoter is believed to contain those elements that regulate thespatio-temporal expression. In addition to the proximal and distalparts, other regulatory regions have also been described, that containenhancer and/or repressors elements The latter elements can be foundfrom a few kilobase pairs upstream from the transcription start site, inthe introns, or even at the 3 side of the genes they regulate (Rombauts,S. et al. (2003) Plant Physiology 132:1162-1176, Nikolov and Burley,(1997) Proc Natl Aced Sci USA 94: 15-22), Tjian and Maniatis (1994) Cell77: 5-8; Fessele et al., 2002 Trends Genet. 18: 60-63, Messing et al.,(1983) Genetic Engineering of Plants; an Agricultural Perspective,Plenum Press, NY, pp 211-227).

When operably linked to a heterologous polynucleotide sequence, apromoter controls the transcription of the linked polynucleotidesequence.

In an embodiment of the present invention, the “cis-actingtranscriptional regulatory elements” from the promoter sequencedisclosed herein can be operably linked to “cis-acting transcriptionalregulatory elements” from any heterologous promoter. Such a chimericpromoter molecule can be engineered to have desired regulatoryproperties. In an embodiment of this invention a fragment of thedisclosed promoter sequence that can act either as a cis-regulatorysequence or a distal-regulatory sequence or as an enhancer sequence or arepressor sequence, may be combined with either a cis-regulatory or adistal regulatory or an enhancer sequence or a repressor sequence or anycombination of any of these from a heterologous promoter sequence.

In a related embodiment, a cis-element of the disclosed promoter mayconfer a particular specificity such as conferring enhanced expressionof operably linked polynucleotide molecules in certain tissues andtherefore is also capable of regulating transcription of operably linkedpolynucleotide molecules. Consequently, any fragment, portion, or regionof the promoter comprising the polynucleotide sequence shown in SEQ IDNO: 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,119, 136 or 139 can be used as a regulatory polynucleotide molecule.

Promoter fragments that comprise regulatory elements can be added, forexample, fused to the 5° end of, or inserted within, another promoterhaving its own partial or complete regulatory sequences (Fluhr et al.,Science 232:1106-1112, 1986; Ellis et al., EMBO J. 6:11-16, 1987;Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990, 1987;Poulsen and Chua, Mol. Gen. Genet. 214:16-23, 1988; Comai et al., PlantMol. Biol. 15:373-381, 1991; 1987; Aryan et al., Mol. Gen. Genet.225:65-71, 1991).

Cis elements can be identified by a number of techniques, includingdeletion analysis, i.e., deleting one or more nucleotides from the 5′end or internal to a promoter; DNA binding protein analysis using DNaseI footprinting; methylation interference; electrophoresis mobility-shiftassays, in vivo genomic footprinting by ligation-mediated PCR; and otherconventional assays; or by sequence similarity with known cis elementmotifs by conventional sequence comparison methods. The fine structureof a cis element can be further studied by mutagenesis (or substitution)of one or more nucleotides or by other conventional methods (see forexample. Methods in Plant Biochemistry and Molecular Biology, Dashek,ed., CRC Press, 1997, pp. 397-422; and Methods in Plant MolecularBiology, Maliga et al., eds., Cold Spring Harbor Press, 1995, pp.233-300).

Cis elements can be obtained by chemical synthesis or by cloning frompromoters that include such elements, and they can be synthesized withadditional flanking sequences that contain useful restriction enzymesites to facilitate subsequent manipulation. Promoter fragments may alsocomprise other regulatory elements such as enhancer domains, which mayfurther be useful for constructing chimeric molecules.

Methods for construction of chimeric and variant promoters of thepresent invention include, but are not limited to, combining controlelements of different promoters or duplicating portions or regions of apromoter (see for example, U.S. Pat. No. 4,990,607; U.S. Pat. No.5,110,732; and U.S. Pat. No. 5,097,025). Those of skill in the art arefamiliar with the standard resource materials that describe specificconditions and procedures for the construction, manipulation, andisolation of macromolecules (e.g., polynucleotide molecules andplasmids), as well as the generation of recombinant organisms and thescreening and isolation of polynucleotide molecules.

In an embodiment of the present invention, the promoters disclosedherein can be modified. Those skilled in the art can create promotersthat have variations in the polynucleotide sequence. The polynucleotidesequence of the promoters of the present invention as shown in SEQ IDNOS: 105-113, 119, 136 or 139, may be modified or altered to enhancetheir control characteristics. As one of ordinary skill in the art willappreciate, modification or alteration of the promoter sequence can alsobe made without substantially affecting the promoter function. Themethods are well known to those of skill in the art. Sequences can bemodified, for example by insertion, deletion, or replacement of templatesequences in a PCR-based DNA modification approach.

The present invention encompasses functional agents and variants of thepromoter sequences disclosed herein.

A “functional fragment” of a regulatory sequence herein is defined asany subset of contiguous nucleotides of any of the regulatory sequencesdisclosed herein, that can perform the same, or substantially similarfunction as the full length promoter sequences disclosed herein.

A “functional fragment of a promoter” with substantially similarfunction to a full length promoter disclosed herein refers to afunctional fragment that retains largely the same level of activity asthe full length promoter sequence and exhibits the same pattern ofexpression as the full length promoter sequence.

A “variant promoter”, as used herein, is the sequence of the promoter orthe sequence of a functional fragment of a promoter containing changesin which one or more nucleotides of the original sequence is deleted,added, and/or substituted, while substantially maintaining promoterfunction. One or more base pairs can be inserted, deleted, orsubstituted internally to a promoter. In the case of a promoterfragment, variant promoters can include changes affecting thetranscription of a minimal promoter to which it is operably linked.Variant promoters can be produced, for example, by standard DNAmutagenesis techniques or by chemically synthesizing the variantpromoter or a portion thereof.

Enhancer sequences refer to the sequences that can increase geneexpression. These sequences can be located upstream, within introns ordownstream of the transcribed region. The transcribed region iscomprised of the exons and the intervening introns, from the promoter tothe transcription termination region. The enhancement of gene expressioncan be through various mechanisms which include, but are not limited to,increasing transcriptional efficiency, stabilization of mature mRNA andtranslational enhancement.

Recombinant DNA constructs of the present invention may also includeother regulatory sequences, including but not limited to, translationleader sequences, introns, and polyadenylation recognition sequences. Inanother embodiment of the present invention, a recombinant DNA constructof the present invention further comprises an enhancer or silencer.

An “intron” is an intervening sequence in a gene that is transcribedinto RNA and then excised in the process of generating the mature mRNA.The term is also used for the excised RNA sequences. An “exon” is aportion of the sequence of a gene that is transcribed and is found inthe mature messenger RNA derived from the gene, and is not necessarily apart of the sequence that encodes the final gene product.

Many genes exhibit enhanced expression on inclusion of an intron in thetranscribed region, especially when the intron is present within thefirst 1 kb of the transcription start site. The increase in geneexpression by presence of an intron can be at both the mRNA (transcriptabundance) and protein levels. The mechanism of this Intron MediatedEnhancement (IME) in plants is not very well known (Rose et al., PlantCell, 20: 543-551 (2008) Le-Hir et al, Trends Biochem Sci. 28: 215-220(2003), Buchman and Berg, Mol. Cell Biol. (1988) 8:4395-4405; Callis etal., Genes Dev. 1 (1987):1183-1200).

An “enhancing intron” is an intronic sequence present within thetranscribed region of a gene which is capable of enhancing expression ofthe gene when compared to an intronless version of an otherwiseidentical gene. An enhancing intronic sequence might also be able to actas an enhancer when located outside the transcribed region of a gene,and can act as a regulator of gene expression independent of position ororientation (Chan et. al. (1999) Proc. Natl. Acad. Sci. 96: 4627-4632;Flodby et al. (2007) Biochem. Blophys. Res. Commun. 356: 26-31).

Short consensus sequences or motifs can be identified from the intronsequences experimentally identified to be enhancing introns. Thesemotifs can be used to scan and help identify more gene-expressionenhancing introns. A motif capable of conferring transgene expression inmale reproductive tissue in dicot plants has been described in USapplication No. US2007/020436.

An 8-bp sequence (SEQ ID NO: 99) and a 5-bp sequence (SEQ ID NO: 100)that can be used for identifying novel enhancing introns have beendescribed in this application. Some variations of the 8-bp sequence canalso be useful for identifying enhancing introns. The useful variationsfrom the 8-bp motif (SEQ ID NO: 99) described herein can occur mainly atthe first three positions. The last 5 bp of the sequence are highlyconserved. Also, the variations from the 8-bp consensus (SEQ ID NO: 99)occur at maximum two out of 8 positions at any one time. In the event ofmore than 2 bp being different than the consensus, the enhancing intronmight have additional copies of either the 5-bp (SEQ ID NO: 100) or the8-bp motif (SEQ ID NO: 99).

The motif variations can be represented as a consensus motif sequence,Y[R/T]RATCYG (SEQ ID NO: 146). The first position can be any of the twopyrimidine bases, C or T. The second position can be substituted by anA, G or T. The third position can be a purine. The ATC core is the mosthighly conserved region, and does not exhibit any variability.

An intron sequence can be added to the 5′ untranslated region, theprotein-coding region or the 3 untranslated region to increase theamount of the mature message that accumulates in the cytosol.

The intron sequences can be operably linked to a promoter. Promoters maybe derived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic DNA segments.

Sequences orthologous to an intron are sequences that are present inorthologous genes at the same position as the intron in the originalgene sequence.

The tissue expression patterns of the genes can be determined using theRNA profile database of the Massively Parallel Signature Sequencing(MPSS™). This proprietary database contains deep RNA profiles of morethan 250 libraries and from a broad set of tissue types. The MPSS™transcript profiling technology is a quantitative expression analysisthat typically involves 1-2 million transcripts per cDNA library(Brenner S. et al., (2000). Nat Biotechnol 18: 630-634, Brenner S. etal. (2000) Proc Natl Acad Sci USA 97: 1665-1670). It produces a 17-basehigh quality usually gene-specific sequence tag usually captured fromthe 3′-most DpnII restriction site in the transcript for each expressedgene. The use of this MPSS data including statistical analyses,replications, etc, has been described previously (Guo M et al. (2008)Plant Mol Biol. 166: 551-563).

IMEter is a word-based discriminator that can do a computationalanalysis as to whether an intron can act as an enhancer of geneexpression or not. The IMeter scoring system is described in Rose, A. B.(2004). Plant J. 40_(—)744-751, and Rose et al (2008) Plant Cell 20:543-551.

“Transcription terminator”, “termination sequences”, or “terminator” asdescribed herein refer to DNA sequences located downstream of a codingsequence, including polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht, I. L., et al., Plant Cell1:671-680 (1989). A polynucleotide sequence with “terminator activity”refers to a polynucleotide sequence that, when operably linked to the 3′end of a second polynucleotide sequence that is to be expressed, iscapable of terminating transcription from the second polynucleotidesequence and facilitating efficient 3′ end processing of the messengerRNA resulting in addition of poly A tail. Transcription termination isthe process by which RNA synthesis by RNA polymerase is stopped and boththe processed messenger RNA and the enzyme are released from the DNAtemplate.

Improper termination of an RNA transcript can affect the stability ofthe RNA, and hence can affect protein expression. Variability oftransgene expression is sometimes attributed to variability oftermination efficiency (Bieri et al (2002) Molecular Breeding 10:107-117). As used herein, the terms “bidirectional transcriptionalterminator” and “bidirectional terminator” refer to a transcriptionterminator sequence that has the capability of terminating transcriptionin both 5″ to 3′, and 3′ to 5′ orientations. A single sequence elementthat acts as a bidirectional transcriptional terminator can terminatetranscription from two convergent genes.

The present invention encompasses functional fragments and variants ofthe terminator sequences disclosed herein.

A “functional fragment of a terminator” with substantially similarfunction to the full length terminator disclosed herein refers to afunctional fragment that retains the ability to terminate transcriptionlargely to the same level as the full length terminator sequence. Arecombinant construct comprising a heterologous polynucleotide operablylinked to a “functional fragment” of the terminator sequence disclosedherein exhibits levels of heterologous polynucleotide expressionsubstantially similar to a recombinant construct comprising aheterologous polynucleotide operably linked to the full lengthterminator sequence.

A “variant terminator”, as used herein, is the sequence of theterminator or the sequence of a functional fragment of a terminatorcontaining changes in which one or more nucleotides of the originalsequence is deleted, added, and/or substituted, while substantiallymaintaining terminator function. One or more base pairs can be inserted,deleted, or substituted internally to a terminator, without affectingits activity. Fragments and variants can be obtained via methods such assite-directed mutagenesis and synthetic construction.

These terminator functional fragments will comprise at least about 20contiguous nucleotides, preferably at least about 50 contiguousnucleotides, more preferably at least about 75 contiguous nucleotides,even more preferably at least about 100 contiguous nucleotides of theparticular terminator nucleotide sequence disclosed herein. Suchfragments may be obtained by use of restriction enzymes to cleave thenaturally occurring terminator nucleotide sequences disclosed herein; bysynthesizing a nucleotide sequence from the naturally occurringterminator DNA sequence; or may be obtained through the use of FORtechnology. See particularly, Mullis et al., Methods Enzymol.155:335-350 (1987), and Higuchi, R. In FOR Technology: Principles andApplications for DNA Amplifications; Erlich, H. A., Ed.; Stockton PressInc.: New York, 1989. Again, variants of these terminator fragments,such as those resulting from site-directed mutagenesis, are encompassedby the compositions of the present invention.

The terms “substantially similar” and “corresponding substantially” asused herein refer to nucleic acid fragments, particularly regulatorysequences, wherein changes in one or more nucleotide bases do notsubstantially alter the ability of the regulatory sequence to performthe same function as the corresponding full length sequence disclosedherein. These terms also refer to modifications, including deletions andvariants, of the nucleic acid sequences of the instant invention by wayof deletion or insertion of one or more nucleotides that do notsubstantially alter the functional properties of the resulting sequencerelative to the initial, unmodified sequence. It is thereforeunderstood, as those skilled in the art will appreciate, that theinvention encompasses more than the specific exemplary sequences.

As will be evident to one of skill in the art, any heterologouspolynucleotide of interest can be operably linked to the regulatorysequences described in the current invention. Examples ofpolynucleotides of interest that can be operably linked to theregulatory sequences described in this invention include, but are notlimited to, polynucleotides comprising other regulatory elements such asintrons, enhancers, promoters, translation leader sequences, proteincoding regions such as disease and insect resistance genes, genesconferring nutritional value, genes conferring yield and heterosisincrease, genes that confer male and/or female sterility, antifungal,antibacterial or antiviral genes, and the like. Likewise, the regulatorysequences described in the current invention can be used to regulatetranscription of any nucleic acid that controls gene expression.Examples of nucleic acids that could be used to control gene expressioninclude, but are not limited to, antisense oligonucleotides, suppressionDNA constructs, or nucleic acids encoding transcription factors.

Embodiments of the invention are:

The present invention relates to regulatory sequences for modulatinggene expression in plants. Recombinant DNA constructs comprisingregulatory sequences are provided. Recombinant DNA constructs comprisingintron sequences acting as enhancers of gene expression and endogenouspromoter and terminator sequences corresponding to these intronsequences are provided.

Another embodiment of the invention is a recombinant DNA constructcomprising an intron operably linked to a promoter and a terminatorwherein the intron comprises a nucleotide sequence that has at least 95%sequence identity to SEQ ID NO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101,102, 103, 104, 118, 137 or 138. In another embodiment, the introncomprises the nucleotide sequence of SEQ ID NO: 4, 8, 13, 19, 52, 53,56, 57, 58, 101, 102, 103, 104, 118, 137 or 138.

One embodiment of the invention is a recombinant DNA constructcomprising an intron operably linked to a promoter and a terminatorwherein the promoter comprises a nucleotide sequence that has at least95% sequence identity to SEQ ID NO: 105-117, 119, 136 or 139. In anotherembodiment, the promoter comprises the nucleotide sequence of SEQ ID NO:105-117, 119, 136 or 139.

One embodiment of the invention is a recombinant DNA constructcomprising an intron operably linked to a promoter and a terminatorwherein the terminator comprises a nucleotide sequence that has at least95% sequence identity to SEQ ID NOS: 140, 141, 142 or 143. In anotherembodiment, the terminator comprises the nucleotide sequence of SEQ IDNO: 140, 141, 142 or 143.

One embodiment of the invention is a recombinant DNA constructcomprising an intron operably linked to a promoter and a terminatorwherein the intron comprises a nucleotide sequence that has at least 95%identity to SEQ ID NOS: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103,104, 118, 137 or 138; and the promoter comprises a nucleotide sequencethat has at least 95% identity to SEQ ID NOS: 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 119, 136 or 139.

One embodiment of the invention is a recombinant DNA constructcomprising an intron operably linked to a promoter and a terminatorwherein the intron comprises a nucleotide sequence that has at least 95%identity to SEQ ID NO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103,104, 118, 137 or 138; the promoter sequence has at least 95% identity toSEQ ID NO: 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 119, 136 or 139; and the terminator has at least 95% sequenceidentity to SEQ ID NO: 140, 141, 142 or 143.

In one embodiment of the current invention, the intron is operablylinked to the promoter, and is present downstream of the promoter, inthe recombinant DNA constructs described herein. One embodiment of thepresent invention includes a recombinant DNA construct comprising anintron described in the present invention, operably linked to a promoterand a heterologous polynucleotide, wherein the intron can act asenhancer of expression of the heterologous polynucleotide.

Another embodiment of the invention encompasses a recombinant DNAconstruct comprising an intron wherein the intron sequence comprises atleast one copy of the 8-bp sequence motif of SEQ ID NO. 99; or containsat least one copy of the 8-bp sequence motif of SEQ ID NO: 99 and atleast one copy of the 5-bp sequence motif of SEQ ID NO: 100, wherein theintron is capable of enhancing expression of a heterologouspolynucleotide in a transgenic plant. The intron sequence can alsocomprise more than one copy of SEQ ID NO: 99, or can comprise one ormore than one copy of SEQ ID NO: 99 and more than one copy of SEQ ID NO:100.

Another embodiment of this invention is a method to identify novelintrons that are useful for enhancing expression of a heterologouspolynucleotide in a plant cell, the method comprising the steps ofscanning a plurality of introns from plants for presence of SEQ ID NO:99, selecting a sequence that contains at least one copy of SEQ ID NO:99, measuring the efficacy of the identified intron to enhanceexpression of a heterologous polynucleotide in a plant.

Another embodiment of the invention is a method for identifying novelintronic sequences for enhancing transgene expression inmonocotyledenous plants by identifying sequences orthologous to SEQ IDNO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103, 104, 118, 137 or138; and measuring the enhancing effect of the identified intron on theexpression of an operably linked heterologous polynucleotide.

Another embodiment of the current invention includes the promoter andthe terminator sequences that are endogenously linked to the intronsidentified using the methods described in the current invention.

Another embodiment of the current invention is a method for modulatingexpression of a heterologous polynucleotide in a monocotyledonous plantcomprising the steps of: (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a promoter and a heterologouspolynucleotide wherein each is operably linked to an intron, wherein theintron comprises either (i) a nucleotide sequence that is orthologous toSEQ ID NO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103, 104, 118,137 or 138; or (ii) a nucleotide sequence that contains least one copyof a sequence motif identical to SEQ ID NO: 99; and; (b) regenerating atransgenic plant from a regenerable monocotyledonous plant cell afterstep (a) wherein the transgenic plant comprises the recombinant DNAconstruct; and (c) obtaining a progeny plant derived from the transgenicplant of step (b), wherein said progeny plant comprises the recombinantDNA construct and exhibits enhanced expression of the heterologouspolynucleotide when compared to a plant comprising a correspondingrecombinant DNA construct without the intron sequence.

In another embodiment, this invention concerns a vector, cell, plant, orseed comprising a recombinant DNA construct comprising the regulatorysequences described in the present invention.

The invention encompasses regenerated, mature and fertile transgenicplants comprising the recombinant DNA constructs described above,transgenic seeds produced therefrom, T1 and subsequent generations. Thetransgenic plant cells, tissues, plants, and seeds may comprise at leastone recombinant DNA construct of interest.

In one embodiment, the plant comprising the regulatory sequencesdescribed in the present invention is a monocotyledenous plant. Inanother embodiment, the plant comprising the regulatory sequencesdescribed in the present invention is a maize plant.

EXAMPLES

The present invention is further illustrated in the following Examples,in which parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these examples,while indicating embodiments of the invention, are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions. Furthermore, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 Identification of Candidate Gene ExpressionTranscript-Enhancing First Introns

Introns that may enhance transcript abundance were sought from among aset of maize genes which (a) had first introns near the N-terminus ofthe transcript, and (b) had high level transcript abundance. A subset ofmaize genes were identified whose models were deemed to be complete.This assessment was done using a combination of maize public B73 BACsequences plus a proprietary EST transcript assembly in an analysiscomparing the predicted gene structures and the predicted transcriptopen reading frames (ORFs) in relation to public reference proteins plussome manual curations. Only full-length transcripts were considered;that is, those with complete protein coding regions. This set did notrepresent all maize genes, and there was some redundancy in the list.

This set of gene models was then analyzed versus a body of over 250 MPSSmRNA transcript profiling samples produced from a variety of maizetissues and treatments. The MPSS profiling technology produces a 17-bptag sequence beginning with GATC. These tags were matched to the geneset via the full-length transcript, and those genes which (a) had anMPSS tag matching the plus strand of the transcript, and (b) had ameasured expression level of at least 1000 ppm (parts per million) in atleast one of the MPSS samples, were retained. In this way a working setof 3131 genes was produced. Using the maize BAC genomic sequence toanalyze these 3131 genes, a subset of genes was produced that (a)contained an intron, and (b) contained an intron which was locatedwithin the 5′UTR or within the first 300 nucleotides of the ORF. Thisresulted in a subset of 1185 genes for further consideration.

This set of 1185 candidate genes was then filtered down by a number ofcriteria. Duplicates were removed. Introns without canonical GT-AG ruleswere excluded. Genes whose expression was defined by ‘promiscuous’ MPSStags, such as GATCAAAAAAAAAAAAA (SEQ ID NO: 145), and also MPSS tagsmatching repetitive elements, were removed. Genes whose first intronswere greater than 2 kb were dropped. In addition, genes whose firstintrons' GC content were higher than 50% GC and/or the intron T (=U)content was below 25% were removed. In addition, the IMeter score forthe first intron had to be positive. The IMeter scoring system isdescribed in Rose, A. B. (2004) Plant J. 40:744-751. This resulted in aninterim set of remaining 331 candidates. This set was then furthermanually winnowed down to 86 by positively considering a combinations offactors but chiefly: (a) the breadth of diverse tissue expression and(b) the ratio of the IMeter score to intron length.

This set of 86 introns was one prioritized pool from which introns weredrawn for functional testing of whether they enhance transcriptabundance. Seventeen of these 86 were tested.

Example 2 Creation of an Intron Testing Vector with Maize UbiquitinPromoter

Maize ubi promoter (SEQ ID NO: 1) along with its intron (SEQ ID NO: 2)in the 5′ UTR confers high level constitutive expression in monocotplants (Christensen, A. H., Sharrock, R. A. and Quail, P. H., Plant Mol.Biol. 18, 675-89, 1992). This high-level expression is dependent on thefirst intron in the 5′ UTR. Removal of this intron results in a >4-foldreduction in expression measured by transient assays (FIG. 3). Wecreated a plant transformation vector where the maize ubiquitin promotertogether with its endogenous intron drives E. coli β-glucuronidase (GUS)reporter gene expression. We then replaced the maize ubiquitin intronwith two restriction sites, AsiS1 and Acc65I to allow the insertion ofnovel introns and test their ability to enhance reporter gene expressiondriven by the ubiquitin promoter (SEQ ID NO: 1) (FIG. 1).

Example 3 Intron Amplification and Cloning

Zea mays B73 seeds were germinated in Petri plates and genomic DNA wasmade from seedling leaf tissue using the QIAGEN® DNEASY® Plant Maxi Kit(QIAGEN® Inc.) according to the manufacturer's instructions. DNAproducts were amplified with primers shown in Table 2 using genomic DNAas template with PHUSION™ DNA polymerase (New England Biolabs Inc.). Theresulting DNA fragments were cloned into the intron testing vectorITVUR-2 (SEQ ID NO: 3), using standard molecular biology techniques(Sambrook et al.) or using INFUSION™ from (Clontech Inc.), and sequencedcompletely.

TABLE 2 Intron Forward Primer Reverse Primer Name SEQ ID NO Length (nt)(SEQ ID NO) (SEQ ID NO) TS1 4 814 20 21 TS4 5 727 22 23 TS5 6 834 24 25TS6 7 982 26 27 TS7v 137 856 28 29 TS8 9 1020 30 31 TS10 10 841 32 33TS11 11 1044 34 35 TS12 12 648 36 37 TS13 13 632 38 39 TS14 14 1405 4041 TS15 15 1361 42 43 TS16 16 703 44 45 TS17 17 1341 46 47 TS24 18 112548 49 TS27v 138 884 50 51

All the constructs were mobilized into the Agrobacterium strainLBA4404/pSB1 and selected on Spectinomycin and tetracycline.Agrobacterium transformants were isolated and the integrity of theplasmid was confirmed by retransforming to E. coli or FOR analysis.

Example 4 Transient Transformation and Expression of Intron Constructsin Maize Embryos Infected with Agrobacterium Preparation ofAgrobacterium Suspension:

Agrobacterium was streaked out from −80° C. frozen aliquot onto a platecontaining PHI-L medium and was cultured at 28° C. in the dark for 2days. The PHI-L medium comprises 50 ml Stock Solution A, 50 ml/L stockSolution B, 900 ml Stock Solution C and spectinomycin (Sigma chemicals)was added to a concentration of 50 mg/L in sterile ddH2O (Stock SolutionA: K2HPO4 60 g/l, NaH2PO4 20 g/l, pH adjusted to 7.0 w/KOH andautoclaved; stock solution B: NH4Cl 20 g/l, MgSO4.7H2O 6 g/l, KCl 3 g/l,CaCl2 0.2 g/l, FeSO4.7H2O 50 mg/l; stock solution C: glucose 5 g/l, agar15 g/l (#A-7049, Sigma Chemicals, St. Louis, Mo.) and was autoclaved.

The plate can be stored at 4° C. and used usually for about 1 month. Asingle colony was picked from the master plate and was streaked onto aplate containing PHI-M medium [Yeast Extract 5 g/l (Difco); Peptone 10g/l (Difco); NaCl 5 g/l (Hi-Media); agar (Sigma Chemicals) 15 g/l; pH6.8, containing 50 mg/l spectinomycin] and incubated at 28° C. in thedark for overnight.

Five ml of PHI-A, [CHU (N6) Basal salts (Sigma 0-1416) 4 g/l; Erikson'svitamin solution (1000×, Sigma-1511) 1 ml/l; Thiamine.HCl (Sigma) 0.5mg/l; 2,4-Dichloro phenoxyacetic acid (2,4-D, Sigma) 1.5 mg/l; L-Proline(Sigma) 0.69 g/l; Sucrose (Sigma) 68.5 μl; Glucose (Sigma) 36 μl; pHadjusted to 5.2 with KOH] was added to a 14 ml FALCON™ tube in a hood.About 3 full loops (5 mm loop size) Agrobacterium was collected from theplate and suspended in the tube, then the tube vortexed to make an evensuspension. One ml of the suspension was transferred to aspectrophotometer tube and the OD of the suspension was adjusted to 0.72at 550 nm by adding either more Agrobacterium or more of the samesuspension medium, for an Agrobacterium concentration of approximately0.5×10⁹ cfu/ml. The final Agrobacterium suspension was aliquoted into 2ml microcentrifuge tubes, each containing 1 ml of the suspension. Thesuspension was then used as soon as possible.

Embryo Isolation, Infection and Co-Cultivation:

About 2 ml of the same medium (PHI-A) which is used for theAgrobacterium suspension was added into a 2 ml microcentrifuge tube.Immature embryos were isolated from a sterilized ear with a sterilespatula and dropped directly into the medium in the tube. A total of 25embryos are placed in the tube. The optimal size of the embryos wasabout 1.7-2.0 mm. The entire medium was drawn off and 1 ml ofAgrobacterium suspension was added to the embryos and the tube wasvortexed for 30 sec. The tube was allowed to stand for 5 min in thehood. The suspension of Agrobacterium and embryos was poured into aPetri plate containing co-cultivation medium PHI-B [CHU(N6) Basal salts(Sigma C-1416) 4 g/l; Eriksson's vitamin solution (1000×, Sigma-1511) 1ml/l; Thiamine.HCl 0.5 mg/l; 2,4-D 1.5 mg/l; L-Proline 0.69 g/l;GELRITE® (Sigma) 3 g/l; Sucrose 30 g/l; pH adjusted to 5.8 with KOH;Post sterilization, Silver nitrate (0.85 mg/l) and acetosyringone (100mM) were added after cooling the medium to 45° C.]. Any embryos left inthe tube were transferred to the plate using a sterile spatula. TheAgrobacterium suspension was drawn off and the embryos placed axis sidedown on the media. The plate was sealed with PARAFILM® and was incubatedin the dark at 23-25° C. for about 3 days of co-cultivation.

Resting of Co-Cultivated Embryos:

For the resting step, all the embryos were transferred to a new platecontaining PHI-C medium [CHU(N6) Basal salts (Sigma C-1416) 4 g/l;Eriksson's vitamin solution (1000×, Sigma-1511) 1 ml/l; Thiamine.HCl 0.5mg/l; 2,4-D 1.5 mg/l; L-Proline 0.69 μl; Sucrose 30 g/l; MES buffer(Sigma) 0.5 g/l; agar (Sigma 1-7049) 8 g/l; pH adjusted to 5.8 with KOH;Post sterilization, Silver nitrate (0.85 mg/l) and carbenicillin (100mg/l) were added after cooling the medium to 45° C.]. The plates weresealed with PARAFILM® and incubated in the dark at 28° C. for 3-5 days.

Histochemical and Fluorometric GUS Analysis:

Transformed embryos were taken for expression analysis after 3 days ofresting. Ten embryos for each construct were used for histochemical GUSstaining using standard protocols (Janssen and Gardner, Plant Mol. Biol.(1989)14:61-72,) and two pools of 5 each were used to do quantitativeassays using MUG substrate using standard protocols [Jefferson, R. A.,Nature. 342:837-838 (1989); Jefferson, R. A., Kavanagh, T. A. & Bevan,M. W. EMBO J. 6:3901-3907 (1987)] (FIG. 3). Introns TS1 (SEQ ID NO: 4),TS7v (SEQ ID NO: 137), TS13 (SEQ ID NO: 13) and TS27v (SEQ ID NO: 138)all enhanced the GUS reporter gene expression between 3 to 5 fold whencompared to the ubiquitin promoter alone without any intron. The levelof enhancement is comparable to that of the maize ubiquitin firstintron. Introns TS4, TS5, TS6, TS8, TS10, TS11, 1512, TS14, TS15, TS16,TS17 and TS24 did not enhance expression (Data shown for TS5, TS6, TS10and T514 in FIG. 3).

Example 5 Transient Transformation and Expression of Intron Constructsin Rice Calli via Agrobacterium Preparation of Agrobacterium Suspension:

Agrobacterium was streaked out from −80° C. frozen aliquot onto a platecontaining YEB medium and was cultured at 28° C. in the dark for 2 days.The YEB medium comprises (MgSO4 (Hi-Media) 0.2 g/l; K2HPO4 (FisherScientific) 0.5 g/l; Mannitol 10 g/l: NaCl 0.1 g/l; Yeast Extract 0.4g/l; Agar 15 g/l). Agrobacterium cultures harboring the intronconstructs were cultured one day prior to rice calli infection in YEBbroth. A large swipe of Agrobacterium growth was inoculated into 7.5 mlof YEB broth in FALCON™ tubes. Then in the next morning OD of eachculture was measured at 550 nm. Cultures were centrifuged at 4000 rpmfor 10 minutes. Supernatant was discarded and the pellet was resuspendedin PHI-L supplemented with Acetosyringone at 100 μM. Another spin wasgiven to Agrobacterium cultures at 4000 rpm for 10 min and the pelletswere resuspended in PHI-L supplemented with Acetosyringone at 100 μM andthe OD was adjusted to 1.0 by adding either more Agrobacterium or moreof the same suspension medium, for an Agrobacterium concentration ofapproximately 0.5×10⁹ cfu/ml.

Rice Callus Induction, Infection and Co-Cultivation

15 to 21 days old Rice calli which were grown on callus inductionmedium, PHI-R [CHU(N6) Basal salts (Sigma C-1416) 40; Eriksson's vitaminsolution (1000×, Sigma-1511) 1 ml/l; Thiamine.HCl 0.5 mg/l: 2,4-D 2.0mg/l; L-Proline 0.69 g/l: Casein hydrolysate (Sigma) 300 mg/l; Sucrose(Sigma) 30 μl; GELRITE® (Sigma) 4 WI; pH adjusted to 5.8 with KOH].Coleoptile of the rice calli was removed and calli were spliced to thesize of approximately 2 to 3 mm. Spliced calli were transferred to theFALCON™ tubes containing Agrobacterium cultures and infected for 15minutes with gentle intermittent shaking. The liquid Agrobacteriumculture was decanted and the wet calli were taken out and blotted onsterile WHATMAN® filter paper No 4. Subsequently, the calli weretransferred onto co-cultivation medium, PHI-R supplemented withAcetosyringone (Sigma) at 100 μM. The infected calli were co-cultivatedin dark at 21° C. for 72 hours.

Resting of Co-Cultivated Rice Calli:

The co-cultivation was terminated by washing in sterile water containingcarbenicillin (Sigma, 400 mg/l). Calli were washed with gentleintermittent shaking in the antibiotic solution for 15 minutes. The wetcalli were blotted on WHATMAN® filter paper No 4. The dried calli weretransferred to resting/callusing medium, PHI-R in which carbenicillin(400 mg/l) was added after cooling the medium to 45° C. aftersterilization. The plates were sealed with PARAFILM® and incubated inthe dark at 28° C. for 3-5 days.

Histochemical and Fluorometric GUS Analysis:

After 3 days, calli were taken for expression analysis. For eachconstruct 20 calli were infected and 8 calli were used for histochemicalGUS staining using X-Gluc solution and another eight calli were takenfor GUS quantitation using standard protocol (Jefferson et al., EMBO J.6:3901-3907, 1987). TS7v (SEQ ID NO: 137) and TS27v (SEQ ID NO: 138)were able to enhance GUS reporter expression from the maize ubiquitinpromoter (SEQ ID NO: 1) (FIG. 4).

Example 6 Description of Constitutive Promoter Selection via MPSSSamples

Promoter candidates were identified using a set of 241 proprietaryexpression profiling experiments run on the MPSS (Massively ParallelSignature Sequencing) technology platform provided by Lynx Therapeutics.The 241 samples from corn consisted of various tissue samples spanningmost of the range of corn tissues and developmental stages. Eachexperiment resulted in approximately 20,000 unique sequence tags of 17bp length from a single tissue sample. Typically these tags could bematched to one or a few transcript sequences from the proprietary“Unicorn” EST assembly set. A query of the MPSS database was performedlooking for tags that were observed in 240 or more of the 241 samples.We identified 111 tags that met the criteria and chose 22 that wereobserved at an expression level of 1 or greater PPM (Parts Per Milliontags) in all 241 experiments for further development. 21 of these 22tags mapped to a single gene based on the transcript set. We took thetop 6 candidates from this list and identified the 1500 bp of promoterregions and the first intron, defined as the first intron in thetranscript from the 5′ end, (i1(SEQ ID NO: 52), i2(SEQ ID NO: 53),i3(SEQ ID NO: 54), i5(SEQ ID NO: 56), i6(SEQ ID NO: 57) and i7(SEQ IDNO: 58). In addition we also included one second intron (i4; SEQ ID NO:55) to the list. All introns were evaluated for intron-mediatedenhancement of expression from CYMV promoter.

Example 7 Enhancement Activity of Introns in Transient Expression System

To determine whether the experimental introns function to enhancepromoter activity in plant tissue, transient infiltration assays usingthe maize suspension cell line, BMS (Black Mexican Sweet), wereperformed. These Agrobacterium-mediated assays, known in the art,provide a rapid screening method to evaluate the enhancement capabilityof the introns.

The introns were cloned into an expression vector downstream of theCitrus Yellow Mosaic virus promoter and upstream of the coding region ofan insecticidal gene described in US2007/0202089 A1. The insecticidalgene acted as a reporter for expression. A vector with no intron betweenthe promoter and coding region was included to provide a baselinecontrol for expression. A vector (SEQ ID NO: 59; PHP38808) with the Adh1intron1 was also included to provide a comparison for the level ofincreased expression by each experimental intron. The Adh1 intron hasbeen shown to enhance the expression of foreign genes in plant tissue(Callis et al. (1987) Genes and Development: 1183-1200; Kyozuka et al.(1990) Maydica 35: 353-357). Each expression vector also contained anexpression cassette for phosphinothricin acetyl transferase (PAT).

Transiently transformed BMS cells were evaluated for expression by bothnorthern blot analysis for RNA accumulation and ELISA analysis forprotein accumulation. If the experimental introns, particularly intronsi1 (SEQ ID NO: 52), i2 (SEQ ID NO: 53), i5 (SEQ ID NO: 56), i6 (SEQ IDNO: 57), and i7 (SEQ ID NO: 58), exhibited intron mediated enhancementof expression, the increased expression would be reflected at both theRNA and protein levels.

The ratio of expression for each intron cassette showed that introns i1,i2, i5, i6, and i7 had expression levels that were between 2.3 and 4.8fold higher than the intronless control (Table 3). These increasedexpression levels were comparable to the control cassette (SEC) ID NO:59, PHP38808; FIG. 5) containing the Adh1 intron. The ELISA values werestandardized for differences in transformation efficiency betweenvectors by normalizing against PAT gene expression.

TABLE 3 ELISA Results Indicating Expression Levels of Insecticidal Gene(IG) and PAT in Constructs Containing Experimental Introns IG PAT IG/Fold difference Intron (ppm) (ppm) PAT from no intron none 38.8 179.00.22 N/A ADH1 104.3 117.4 0.89 4.05 i1 98.3 136.5 0.72 3.27 i2 118.7154.0 0.77 3.50 i5 115.5 108.5 1.06 4.82 i6 107.6 209.0 0.51 2.32 i7104.3 117.4 0.89 4.05

To determine whether introns i1 (SEQ ID NO: 52), i2 (SEQ ID NO: 53), i5(SEQ ID NO: 56), i6 (SEQ ID NO: 57), and i7 (SEQ ID NO: 58) resulted inincreased mRNA levels, northern blot analysis was performed. RNA amountsfor each vector were normalized against PAT expression prior toelectrophoresis. The results of the analysis mirrored the ELISA results.Introns i1, i2, i5, i6, and i7 facilitated levels of reporter mRNAaccumulation that were above that of the intronless cassette andcomparable to the ADH1 cassette (see FIG. 6). These results show thati1, i2, i5, i6, and i7 (SEQ ID NOS: 52-53, 56-58 respectively) displayintron-mediated enhancement of expression in this system.

Materials and Methods:

Introns i1 (SEQ ID NO: 52), i2 (SEQ ID NO: 53), i3 (SEQ ID NO: 54), i4(SEQ ID NO: 55) and i5 (SEQ ID NO: 56) were generated using a methodknown in the art as oligonucleotide stacking. Oligos and primers (Table4) synthesized by IDT (Integrated DNA Technologies, Inc. Coralville,Iowa) were resuspended in distilled water to a concentration of 100 μM.Equal amounts of each oligonucleotide were mixed to create a totalvolume of 10 μl. The flanking primers for PCR amplification were alsomixed equally to a volume of 10 μl. Two microliters of theoligonucleotide mix and 10 μl of the primer mix were combined for PCRusing the HotStart Herculase system from Stratagene. PCR was performedusing 10 μl Herculase buffer, 20 of 25 nM dNTPs, 1.2 μl of the oligo andprimer mixture, 1 μl 100 mM MgSO4, 2 μl DMSO, 1 μl HotStart Herculaseenzyme, and 82.8 μl of distilled water. PCR conditions were 96° C. for 3minutes, then 35 cycles at 94° C. for 30 s, 60° C. for 30 s, and 72° C.for 1 min., followed by 72° C. for 10 min. Reactions were stored at 4°C. Introns i6 and i7 were synthesized by GENEART, Inc., Burlingame,Calif. To clone introns i1 (SEQ ID NO: 52), i2 (SEQ ID NO: 53), i6 (SEQID NO: 57), and i7 (SEQ ID NO: 58), the starting product was cut withthe restriction enzymes ECoRV (5′ end) and BamHIH (3′ end). Intron i5(SEQ ID NO: 56), was cut with EcoRV (5′ end) and BglII (3′ end). Aplasmid containing a cassette (SEQ ID NO: 59, PHP38808; FIG. 5) with theCYMV promoter the ADH1 intron and an insecticidal gene flanked byGATEWAY® (INVITROGENT™) attL recombination sites was cut with EcoRV andBamHI to remove the ADH1 intron and allow the experimental introns to beligated into the cut plasmid. The resulting vectors (entry vectors,PHP38811 PHP38813, PHP38815, PHP38817, PHP38819, PHP38821 PHP38823 fori2, i3 i4, i5, i6, i7 respectively) were used in LR reactions with alarger plasmid (PHP34651, FIG. 7, SEQ ID NO: 60) containing GATEWAY®attR recombination sites and a PAT expression cassette to generate thefinal expression vectors (destination vectors PHP38812, PHP38814,PHP38816, PHP38818, PHP38820, PHP38822 and PHP38824 respectively forintrons i1, i2, i3, i4, i5, i6, i7, i8 and i9). These vectors were usedto transform competent Agrobacterium tumefaciens cells, which were thenused to transiently transform BMS cells,

TABLE 4 Primers and Oligonucleotides Used for Oligonucleotide StackingOligo/Primer (Used for) Sense/ Flanking SEQ ID NO: Intron AntisensePrimer/Oligonucleotide 61 i1 Sense Flanking Primer 62 i1 SenseOligonucleotide 63 i1 Sense Oligonucleotide 64 i1 Sense Oligonucleotide65 i1 Antisense Oligonucleotide 66 i1 Antisense Oligonucleotide 67 i1Antisense Oligonucleotide 68 i1 Antisense Flanking Primer 69 i2 SenseFlanking Primer 70 i2 Sense Oligonucleotide 71 i2 Sense Oligonucleotide72 i2 Sense Oligonucleotide 73 i2 Antisense Oligonucleotide 74 i2Antisense Oligonucleotide 75 i2 Antisense Oligonucleotide 76 i2Antisense Flanking Primer 77 i3 Sense Flanking Primer 78 i3 SenseOligonucleotide 79 i3 Sense Oligonucleotide 80 i3 AntisenseOligonucleotide 81 i3 Antisense Oligonucleotide 82 i3 Antisense FlankingPrimer 83 i4 Sense Flanking Primer 84 i4 Sense Oligonucleotide 85 i4Sense Oligonucleotide 86 i4 Antisense Oligonucleotide 87 i4 AntisenseOligonucleotide 88 i4 Antisense Flanking Primer 89 i5 Sense FlankingPrimer 90 i5 Sense Oligonucleotide 91 i5 Sense Oligonucleotide 92 i5Antisense Oligonucleotide 93 i5 Antisense Oligonucleotide 94 i5Antisense Flanking Primer

RNA was extracted from infiltrated tissue culture material using the(DAGEN® RNA Maxiprep kit. Based on ELISA data for PAT, RNA samples wereloaded on an agarose gel (1% Lonza SeaKem LE agarose) to contain equalparts per million of PAT to normalize for variations in transformationefficiency. After electrophoresis, samples on the gel were transferredto a nylon membrane via capillary transfer overnight using the WHATMAN®TurboBlotter system standard protocol. RNA was crosslinked to themembrane by UV light. Prehybridization and hybridization steps wereperformed following the manufacturer's protocol for Roche DIG Easy Hybsolution (catalog #11603558001). The blot was prehybridized at 50′C inRoche DIG Easy Hyb solution, then was probed overnight at 50′C with amixture of digoxigenin-labeled DNA probes for the insecticidal and PATgene in Roche DIG Easy Hyb solution. Probes were generated using RochePCR DIG Probe Synthesis Kit (Roche catalog #11636090910). The blot waswashed twice for five minutes each at room temperature in low stringencybuffer (2×SSC+0.1% SDS), then washed twice for 15 minutes each at 50′Cin high stringency buffer (0.1×SSC+0.1% SDS).

For detection, the Roche DIG Wash and Block Buffer Set (catalog#11585762001) was used. The membrane was washed for 2 minutes at roomtemperature in wash buffer, and then blocked in block solution for 30minutes at room temperature. A 1:10,000 dilution of anti-digoxigenin-APantibody (Roche catalog #11093274910, 0.75 U/μl) in 50 ml block solutionwas added to the blot for 30 minutes. The blot was washed twice for 15minutes each at room temperature in wash buffer, and then equilibratedin 50 ml of detection buffer for 3 minutes. Blot was incubated at roomtemperature for 5 minutes with 3 ml of CSPD (Roche catalog #1755633001),and then incubated at 37° C. for 10 minutes. Detection was done withfilm at 37° C.

Example 8 Identification of Unique Motif from Maize First Introns Usingthe Experimental Dataset of Tested Enhancing Introns

Computational analysis was performed to identify unique motifs that werepresent in the 9 enhancing introns identified as explained in Examples 4and 7 and Table 1 (TS1, TS7, TS13, TS27, i1, i2, i5, i6, i7(SEQ ID NOS:4, 8, 13, 19, 52, 53, 56, 57, and 58 respectively)). The proprietarypromoter REAPer tool was adapted to look for possibly conserved motifs.The promoter REAPer tool is a regulatory element identification toolthat relies on the conserved word approach. It is described in the U.S.patent application Ser. No. 12/534,471. The introns were searched inboth directions using sets of 3-6 introns at a time. When candidateswere found, they were used to search all the introns.

The introns were divided into the following categories. “All EnhancingIntrons” are the 9 introns (new enhancing introns) described in Table 1and experimentally shown to be enhancing gene expression (TS1, TS7,TS13, TS27, i1, i2, i5, i6, and i7 (SEQ ID NOS: 4, 8, 13, 19, 52, 53,56, 57, and 58 respectively), plus four known enhancing introns(Adh1_intron1 (SEQ ID NO: 95), Adh1_intron 6 (SEQ ID NO: 96),Sh-1_intron 1 (SEQ ID NO: 97) and Ubi1ZM_intron (SEQ ID NO: 98) Callis,J. et al (1987) Genes Dev. 1: 1183-1200, Vasil, V. et al (1989) PlantPhysiol. 91; 1575-1579, Christensen, A. H. et al (1992) Plant Mol. Biol.18: 675-689, Jeong, Y.-M. at al (2009) Plant Sci. 176:58-65). The 10“non-enhancing introns” are 10 introns found not to enhance geneexpression in transient maize assays as explained in Examples 4 and 7and Table 1 (SEQ ID NOS: 5-7, 9, 11, 12, 17, 18, 54, and 55).

The 8-bp sequence CAGATCTG (SEQ ID NO: 99) or its variations were foundin all the enhancing introns except TS27. The exact 8-bp sequenceCAGATCTG was found in 2 out of the 9 enhancing introns identified (SEQID NOS: 52 and 53), but was not found in any of the 10 non-enhancingintrons (SEQ ID NOS: 5-79, 11, 12, 17, 18, 54, and 55). A subset of thissequence ATCTG (SEQ ID NO: 100) was also present in 8 out of 9 enhancingintrons (SEQ ID NOS: 4, 8, 13, 52, 53, 56, 57 and 58), and was alsofound to be present in the four known enhancing introns (SEQ ID NOS:95-98). The frequency of occurrence of these motifs was normalized tothe intron length (Table 6).

The variations of the 8-bp sequence CAGATCTG are mainly in the first 3base pairs. The motif variations can be represented as the consensussequence, Y[R/T]RATCYG (SEQ ID NO: 146). The first position can be anyof the two pyrimidine bases, C or T. The second position can besubstituted by an A, G or T and the third position can any purine. Thelast 5 base pairs of the sequence, that is the sequence ATCTG is highlyconserved.

Statistical Analyses of Motif Frequencies:

A number of simple frequency statistics were determined for the introns.The statistics are shown in Tables 5 and 6.

TABLE 5 Aggregate Average Intron Intron Classification Intron Count NtsLength All Enhancing Introns 13 7716 594 New Enhancing Introns 9 4813535 Other Enhancing Introns 4 2903 726 Non-Enhancing Introns 10 7888 789Non-Tested Introns 1066 933097 875

TABLE 6 Total Introns Total Introns Frequency Frequency ContainingContaining Intron Contains Intron Contains Intron ClassificationCAGATCTG ATCTG CAGATCTG ATCTG All Enhancing Introns 2 12 0.15 0.92 NewEnhancing Introns 2 8 0.22 0.89 Other Enhancing Introns 0 4 0.00 1.00Non-Enhancing Introns 0 7 0.00 0.70 Non-Tested Introns 15 502 0.01 0.47Ratio All Enhancing/ 1.71 1.32 Non-Enhancing Ratio New Enhancing/ 1.141.27 Non-Enhancing Total Total Occurrences Occurrences Gross GrossCAGATCTG ATCTG Frequency Frequency Intron Classification Either StrandEither Strand CAGATCTG ATCTG All Enhancing Introns 6 29 0.0008 0.0038New Enhancing Introns 6 23 0.0012 0.0048 Other Enhancing Introns 0 6 00.00207 Non-Enhancing Introns 0 18 0 0.00228 Non-Tested Introns 15 13911.6075E−05 0.00149 Ratio All Enhancing/ 1.61 1.647 Non-Enhancing RatioNew Enhancing/ 1.28 2.094 Non-Enhancing Average of Average IndividualIndividual Intron SE SE Frequency of Frequency of Frequency FrequencyIntron Classification CAGATCTG/kb ATCTG/kb CAGATCTG/kb ATCTG/kb AllEnhancing Introns 0.0036 0.0094 0.0025 0.0004 New Enhancing Introns0.0052 0.0124 0.0035 0.0050 Other Enhancing Introns 0.00000 0.002660.00000 0.00107 Non-Enhancing Introns 0.00000 0.00203 0.00000 0.00057Non-Tested Introns 0.00013 0.00271 0.00005 0.00013 Ratio All Enhancing/4.62 Non-Enhancing Ratio New Enhancing/ 6.10 Non-Enhancing

SE frequency is standard error of frequency. Gross frequency is simplythe total occurrences divided by the aggregate nucleotides of all theintrons in the set.

The ‘all’ 13 enhancing introns have 4.6-fold higher, and the 9 ‘new’enhancing introns have 6.1-fold higher frequencies of ATCTG relative tothe non-enhancing introns on a mean frequency per kb of intron basis(See Tables 5 and 6 above).

Example 9 Identification of Novel Maize Introns with 8-b Motif

From the initial set of 1085 introns explained in Example 1, 1066introns that were still not tested experimentally were scannedcomputationally to identify the ones with the 8-bp motif. Four introns(SEQ ID NOS: 101-104) were found to contain the exact 8-bp motif andthese are good candidates for being enhancing introns.

Example 10 Identifying Promoters of Expression-Enhancing Introns

It is likely that the expression enhancing introns from Examples 4, 7and 9 perform optimally along with their endogenous promoters. To testthis 1000 bp-2000 bp of promoter regions upstream of the start codonfrom the respective genes (SEQ ID NOS: 105-117, SEQ ID NOS: 136 and 139)were identified and these can be tested with the respective introns.

Cloning Endogenous Promoters of Expression Enhancing Introns

We amplified 1000 base pairs region of endogenous promoter, (using theprimers given in Table 7) upstream of the start codon of the gene thatcarries TS1 intron as its first intron and cloned the pTS1v sequence(SEQ ID NO: 136) in ITVUR-2 vector (SEQ ID NO: 3, PHP41353) betweenAscI-AsiS1 restriction sites, followed by the TS1 intron (SEQ ID NO: 4)at AsiSI-Acc65I sites to create an endogenous promoter and introncombination (PHP50061). Similarly, we amplified a 1487 base pair regionof endogenous promoter (pTS27v: SEQ ID NO: 139) upstream of the TS27intron and cloned it in ITVUR-2 vector (SEQ ID NO: 3, PHP41353) atAscI-AsiS1 restriction sites, followed by the TS27v intron (SEQ ID NO:138) at AsiSI-Acc651 sites to give us an endogenous promoter and introncombination (PHP52322).

Example 11 Cloning and Testing of TS2 Enhancing Intron and CorrespondingEndogenous Promoter

We tested another intron with potential gene expression enhancingproperties. TS2 intron (SEQ ID NO: 118) was cloned into ITVUR-2 vector(SEQ ID NO: 3, PHP41353) using the same procedure as explained inExample 3 to create PHP50062. We created 2 more constructs to test theability of the endogenous promoter upstream of the start codon of thegene that carries TS2 as its first intron to drive gene expression andability of TS2 intron to enhance gene expression. We amplified 1077-bpof endogenous TS2 promoter (pTS2; SEQ ID NO: 119), as defined by thesequence upstream of the TS2 intron at the genomic location, and clonedthat in ITVUR-2 vector (SEQ ID NO: 3) between AscI and NcoI sites(PHP500063). We also amplified the pTS2 promoter and TS2 intron sequencefrom the endogenous locus (1077 bp promoter (SEQ ID NO: 118)+1329 bpintron (SEQ ID NO: 119)) and cloned that between AscI and NcoI sites(PHP50111), The primers for these amplifying promoter and intronsequences to make these constructs are given in Table 2 and Table 7.

TABLE 7 Cloned sequence Forward Primer Reverse Primer Promoter Intron(SEQ ID NO) (SEQ ID NO) — TS2 (SEQ ID 120 121 NO: 118) pTS2 (SEQ ID TS2(SEQ ID 122 123 NO: 119) NO: 118) pTS2 (SEQ ID — 122 124 NO: 119) pTS1v(SEQ ID — 125 126 NO: 136) pTS27v (SEQ — 127 128 ID NO: 139)

All the constructs were mobilized into the Agrobacterium strainLBA4404/pSB1 and selected on spectinomycin and tetracycline.Agrobacterium transformants were isolated and the integrity of theplasmid was confirmed by retransforming to E. coli or PCR analysis.

Example 12 Stable Transfection of Rice with Promoter and Intron SequenceConstructs Transformation and Regeneration of Rice Callus viaArobacterium Infection

O. saliva spp. japonica rice var. Nipponbare seeds are sterilized inabsolute ethanol for 10 minutes then washed 3 times with water andincubated in 70% Sodium hypochlorite [Fisher Scientific-27908] for 30minutes. The seeds are then washed 5 times with water and driedcompletely. The dried seeds are inoculated into NB-CL media [CHU(N6)basal salts (PhytoTechnology-0416) 4 g/l; Eriksson's vitamin solution(1000× PhytoTechnology-E330) 1 ml/l; Thiamine HCl (Sigma-T4625) 0.5mg/l; 2,4-Dichloro phenoxyacetic acid (Sigma-D7299) 2.5 mg/l; BAP(Sigma-B3408) 0.1 mg/l; L-Proline (PhytoTechnology-P698) 2.5 g/l; Caseinacid hydrolysate vitamin free (Sigma-C7970) 0.3 g/l; Myo-inositol(Sigma-13011) 0.1 μl; Sucrose (Sigma-S5390) 30 μl; GELRITE®(Sigma-G1101.5000) 3 g/l; pH 5.8) and kept at 28° C. in dark for callusproliferation.

A single Agrobacterium colony containing a desired insert with thecandidate sequences from a freshly streaked plate can be inoculated inYEB liquid media [Yeast extract (BD Difco-212750) 1 g/l; Peptone (BDDifco-211677) 5 g/l; Beef extract (Amresco-0114) 5 g/l; Sucrose(Sigma-S5390) 5 g/l; Magnesium Sulfate (Sigma-M8150) 0.3 g/l at pH-7.0]supplemented with Tetracycline (Sigma-T3383) 5 mg/l, Rifamysin 10 mg/land Spectinomycin (Sigma-5650) 50 mg/l. The cultures are grown overnightat 28° C. in dark with continuous shaking at 220 rpm. The following daythe cultures are adjusted to 0.5 Absorbance at 550 nm in PHI-A(CHU(N6)basal salts (PhytoTechnology-C416) 4 g/l; Eriksson's vitamin solution(1000× PhytoTechnology-E330) 1 ml/l; Thiamine HCl (Sigma-T4625) 0.5mg/l; 2,4-Dichloro phenoxyacetic acid (Sigma-D7299) 2.5 mg/l, L-Proline(PhytoTechnology-P698) 0.69 mg/l; Sucrose (Sigma-S5390) 68.5 g/l;Glucose-36 g/l (Sigma-G8270); pH 5.8);) media supplemented with 200 μMAcetosyringone (Sigma-D134406) and incubated for 1 hour at 28° C. withcontinuous shaking at 220 rpm.

17-21 day old proliferating calli are transferred to a sterile cultureflask and Agrobacterium solution prepared as described above was addedto the flask. The suspension is incubated for 20 minutes with gentleshaking every 2 minutes. The Agrobacterium suspension is decantedcarefully and the calli are placed on WHATMAN filter paper No-4. Thecalli are immediately transferred to NB-CC medium [NB-CL supplementedwith 200 μM Acetosyringone (Sigma-D134406) and incubated at 21° C. for72 hrs.

Culture Termination and Selection

The co-cultivated Calli are placed in a dry, sterile, culture flask andwashed with 1 liter of sterile distilled water containing Cefotaxime(Duchefa-00111.0025) 0.250 μl and Carbenicillin (Sigma-00109.0025) 0.4μl. The washes are repeated 4 times or until the solution appearedclear. The water is decanted carefully and the calli are placed onWHATMAN filter paper No-4 and dried for 30 minutes at room temperature.The dried calli are transferred to NB-RS medium [NB-CL supplemented withCefotaxime (Duchefa-00111.0025) 0.25 WI; and Carbenicillin(Sigma-00109.0025) 0.4 g/l and incubated at 28° C. for 4 days.

The calli are then transferred to NB-SB media [NB-RS supplemented withBialaphos (Meiji Seika K.K., Tokyo, Japan) 5 mg/l and incubated at 28°C. and subcultured into fresh medium every 14 days. After 40-45 days onselection, proliferating, Bialaphos resistant, callus events are easilyobservable.

Regeneration of Stably Transformed Rice Plants from Transformed RiceCalli

Transformed callus events are transferred to NB-RG media [CHU(N6) basalsalts (PhytoTechnology-C416) 4 g/l; N6 vitamins 1000×1 ml {Glycine(Sigma-47126) 2 g/l; Thiamine HCl (Sigma-T4625) 1 WI; acid; Kinetin(Sigma-K0753) 0.5 mg/l; Casein acid hydrolysate vitamin free(Sigma-C7970) 0.5 μl; Sucrose (Sigma-S5390) 20 g/l; Sorbitol(Sigma-S1876) 30 g/l, pH was adjusted to 5.8 and 4 WI GELRITE®(Sigma-G1101,5000) was added. Post-sterilization 0.1 ml/l of CuSo4 (100mM concentration, Sigma-C8027) and 100 ml/l 10× AA Amino acids pH free{Glycine (Sigma-G7126) 75 mg/l; L-Aspartic acid (Sigma-A9256) 2.66 g/l;L-Arginine (Sigma-A5006) 1.74 WI; L-Glutamine (Sigma-G3126) 8.76 g/l}and incubated at 32° C. in light. After 15-20 days, regeneratingplantlets can be transferred to magenta boxes or tubes containing NB-RTmedia [MS basal salts (PhytoTechnology-M524) 4.33 g/L; B5 vitamins 1ml/l from 1000× stock {Nicotinic acid (Sigma-G7126) 1 g/l, Thiamine HCl(Sigma-T4625) 10 g/1)}: Myo-inositol (Sigma-13011) 0.1 g/l; Sucrose(Sigma-S5390) 30 μl; and IBA (Sigma-15386) 0.2 mg/l; pH adjusted to5.8]. Rooted plants obtained after 10-15 days can be hardened in liquidY media [1.25 ml each of stocks A-F and water sufficient to make 1000ml. Composition of individual stock solutions: Stock (A) AmmoniumNitrate (HIMEDIA-RM5657) 9.14 μl, (B) Sodium hydrogen Phosphate(HIMEDIA-58282) 4.03 g, (C) Potassium Sulphate (HIMEDIA-29658-4B) 7.14g, (D) Calcium Chloride (HIMEDIA-05080) 8.86 g, (E) Magnesium Sulphate(HIMEDIA-RM683) 3.24 g, (F) (Trace elements) Magnesium chloride tetrahydrate (HIMEDIA-10149) 15 mg, Ammonium Molybdate (HIMEDIA-271974B) 6.74mg/l, Boric acid (Sigma-136768) 9.34 g/l, Zinc sulphate heptahydrate(HiMedia-RM695) 0.35 mg/l, Copper Sulphate heptahydrate (HIMEDIA-08027)0.31 mg/l, Ferric chloride hexahydrate (Sigma-236489) 0.77 mg/l, Citricacid monohydrate (HIMEDIA-04540) 0.119 g/l] at 28° C. for 10-15 daysbefore transferring to greenhouse. Leaf samples are collected forhistochemical GUS staining with5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), using standardprotocols (Janssen and Gardner, Plant Mol. Biol. (1989)14:61-72).

Transgenic plants are analyzed for copy number by southern blottingusing standard procedure. All single copy events are transferred toindividual pots and further analysis is performed only on these. For allthe analysis leaf material from three independent one month old singlecopy T₀ events were taken.

Transgene Copy Number Determination by Quantitative FOR

Transgenic rice plants generated using different constructs wereanalyzed to determine the transgene copy number using TaqMan-basedquantitative real-time FOR (qPCR) analysis. Genomic DNA was isolatedfrom the leaf tissues collected from 10-day old T0 rice plants using theQIAGEN® DNEASY® Plant Maxi Kit (QIAGEN® Inc.) according to themanufacturers instructions. DNA concentration was adjusted to 100 ng/μland was used as a template for the qPCR reaction to determine the copynumber. The copy number analysis was carried out by designing PCRprimers and TaqMan probes for the target gene and for the endogenousglutathione reductase 5 (GR5) gene. The endogenous GR5 gene serves as aninternal control to normalize the Ct values obtained for the target geneacross different samples. In order to determine the relativequantification (RQ) values for the target gene, genomic DNA from knownsingle and two copy calibrators for a given gene were also included inthe experiment. Test samples and calibrators were replicated twice foraccuracy. Non-transgenic control and no template control were alsoincluded in the reaction. The reaction mixture (for a 20 μl reactionvolume) comprises 10 μl of 2× TagMan universal FOR master mix (AppliedBiosystems), 0.5 μl of 10 μM FOR primers and 0.5 μl of 10 μM TaqManprobe for both target gene and endogenous gene. Volume was adjusted to19 μl using sterile Milli Q water and the reaction components were mixedproperly and spun down quickly to bring the liquid to bottom of thetube. 19 μl of the reaction mix was added into each well of reactionplate containing 1 μl of genomic DNA to achieve a final volume of 20 μl.The plate was sealed properly using MicroAmp optical adhesive tape(Applied Biosystems) and centrifuged briefly before loading onto theReal time FOR system (7500 Real FOR system, Applied Biosystems). Theamplification program used was: 1 cycle each of 50° C. for 2:00 min and95° C. for 10:00 min followed by 40 repetitions of 95° C. for 15 sec and58° C. for 1:00 min. After completion of the FOR reaction, the SDS v2.1software (Applied Biosystems) was used to calculate the RQ values in thetest samples with reference to single copy calibrator.

Stable transgenic rice events were generated with the constructs,PHP50063, PHP50111 PHP50062, PHP50061, PHP52322, and PHP42365 as givenin Table 8. The primers used for amplifying the cloned promoter andintron sequences for these constructs are given in Table 2 and Table 7.

TABLE 8 Description of Promoter and Intron Elements in ConstructsConstruct Intron Promoter PHP50063 — pTS2 (SEQ ID NO: 119) PHP50111 TS2pTS2 (SEQ ID NO: 118) (SEQ ID NO: 119) PHP50062 TS2 Zm Ubi promoter (SEQID NO: 118) PHP50061 TS1 pTS1v (SEQ ID NO: 4) (SEQ ID NO: 136) PHP52322TS27v pTS27v (SEQ ID NO: 138) (SEQ ID NO: 139) PHP42365 Zm Ubi intron ZmUbi promoter

The stable transgenic rice events generated with these constructs weresubjected to TaqMan-based qPCR (quantitative PCR) analysis to determinethe transgene copy number as described above. FOR primers and TagManprobes designed for the GUS reporter gene and for the endogenous GR5gene are listed in Table 9.

TABLE 9 Primer Sequences for qPCR Primer ID SEQ ID NO: GUS F primer 129GUS R primer 130 GR5, F primer 131 GR5, R primer 132

TABLE 10 Probe Sequences for qPCR SEQ ID NO Probe Quencher GUS 133 FamTamra GR5 134 Vic MGB

All single copy events were transferred to individual pots and furtheranalysis was performed on leaf material and panicle collected one monthafter transplanting in the greenhouse.

Qualitative and Quantitative Analysis of GUS Reporter Gene Expression inStable Rice Events

Both qualitative and quantitative GUS reporter gene expression analyseswere carried out in triplicates on at least 5 independent single copyevents for each construct. Leaf and panicle samples were collected forhistochemical GUS staining with5-brorno-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), using standardprotocols (Janssen and Gardner, Plant Mol. Biol. (1989)14:61-72) and forquantitative MUG assay using standard protocols (Jefferson, R. A.,Nature. 342, 837-8 (1989); Jefferson, R. A., Kavanagh, T. A. & Bevan, M.W., EMBO J. 6, 3901-3907 (1987).

TS1 and TS27v when combined with their respective endogenous promoters(pTS1v+TS1 (PHP50061) and pTS27v+TS27v (PHP52322) were able to drive GUSexpression in stable rice transgenic events (FIG. 9).

TS2 intron with its endogenous promoter (PHP50111) enhanced the GUSreporter gene expression by 11.6 fold in leaves and 8.9 fold in paniclescompared to the TS2 promoter alone (PHP50063) driving the GUS reportergene expression (FIG. 10) and the values obtained were comparable to thelevels observed with maize ubiquitin promoter and intron (PHP42365)driving GUS in transgenic rice plants. There is a slight increase in theGUS reporter gene expression levels when the TS2 intron is cloned withmaize Ubiquitin promoter (PHP50062) compared to the data obtained withmaize ubiquitin intron cloned with maize ubiquitin promoter (FIG. 10).

GUS histochemical staining data were found to correlate very well withthe quantitative GUS assay in all events. Representative images areshown in FIG. 10 and FIG. 11.

Example 13 Identification of Novel Terminator Sequences

Transcription terminators for the 4 genes comprising the expressionenhancing introns TS1, TS2, TS13 and TS27v (SEQ ID NOS: 4, 118, 13 and138 respectively) were identified, and were called tTS1 (SEQ ID NO:140), tTS2 (SEQ ID NO: 141), tTS13 (SEQ ID NO: 142) and tTS27 (SEQ IDNO: 143). Terminator sequences were defined as 500-900 bp of sequencedownstream of the translational stop codon of the respective genes.

Example 14 Amplification and Cloning of Terminator Sequences

We constructed a terminator test vector (TTV) (PHP49597-FIG. 13; SEQ IDNO: 144) carrying GUS (β-glucuronidase) reporter gene driven by theMaize Ubiquitin promoter using standard molecular biology techniques(Sambrook et al.), A promoterless Ds-RED coding sequence was includeddownstream of the GUS gene for measurement of transcription downstreamof the cloned test terminator sequences (read-through transcripts). TheDs-Red sequence was followed by a PinII terminator to enable terminationand polyadenlylation of all transcripts, so we could detect them byreverse-transcription-PCR (RT-PCR) using oligo dT primer. The Terminatortest vector also carried a monocot-optimized Phosphinothricin acetyltransferase (MOPAT) gene as a plant selectable marker.

Candidate terminator sequences can be amplified from maize genomic DNA.The resulting DNA fragments can be cloned into the terminator testvector at Acc65I restriction site using IN-FUSION™ cloning (ClontechInc.). All constructs will be transformed into Agrobacterium(LBA4404/pSB1)

Example 15 Rice Transformation with Candidate Terminator Sequences

The candidate maize terminator sequences tTS1, tTS2, tTS13 and tTS27(SEQ ID NOS:140-143 respectively) will be tested for their ability tofunction as transcription terminators in stable rice transgenic plantsgenerated by Agrobacterium mediated transformation as described inExample 12.

Example 16 Testing of Candidate Rice Terminator Sequences in StablyTransformed Rice Tissues

Reverse Transcriptase-PCR (RT-PCR) and GUS assays can be done fromstably transformed rice plant tissues, to test the ability of candidatemaize terminator sequences tTS1, tTS2, tTS13 and tTS27 (SEC) ID NOS:140-143 respectively) to prevent transcription read-through and tocompare GUS expression

Reverse Transcription PCR (RT-PCR) to Determine TranscriptionRead-Through

RNA will be extracted from leaf tissue from multiple independent T0events for each construct. cDNA can be synthesized using SuperScript®III First-Strand Synthesis System from Invitrogen. The level of GUS geneand read-through transcripts will be assayed using specific primerswithin GUS gene and DS-Red respectively. Transcript levels can also bemeasured by quantitative RT-PCR using primers and probes within GUS andDS-Red sequences.

Histochemical and Fluorometric GUS Analysis

Tissue samples from each independent stably transformed rice line can bestained for histochemical GUS analysis, with5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), using standardprotocols (Janssen and Gardner, Plant Mol. Biol. (1989)14:61-72,).Tissue samples can also be used for quantitative MUG assay usingstandard protocols [Jefferson, R. A., Nature. 342:837-838 (1989);Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. EMBO J. 6:3901-3907(1987)].

1. A recombinant DNA construct comprising an intron operably linked to apromoter, a heteroloqous polynucleotide and a terminator wherein theintron comprises a nucleotide sequence that has at least 95% sequenceidentity to SEQ ID NO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103,104, 118, 137 or
 138. 2. (canceled)
 3. (canceled)
 4. The recombinant DNAconstruct of claim 1, wherein the promoter comprises a nucleotidesequence that has at least 95% identity to SEQ ID NO: 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 119, 136 or
 139. 5.The recombinant DNA construct of claim 4, wherein the terminatorcomprises a nucleotide sequence that has at least 95% identity to SEQ IDNO: 140, 141, 142 or
 143. 6. The recombinant DNA construct of claim 1,wherein the intron comprises the nucleotide sequence of SEQ ID NO: 4, 8,13, 19, 52, 53, 56, 57, 58, 101, 102, 103, 104, 118, 137 or
 138. 7. Therecombinant DNA construct of claim 4, wherein the promoter comprises thenucleotide sequence of SEQ ID NO: 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 119, 136 or
 139. 8. The recombinant DNAconstruct of claim 5, wherein the terminator comprises the nucleotidesequence of SEQ ID NO: 140, 141, 142 or
 143. 9. (canceled)
 10. Therecombinant DNA construct of claim 1, wherein the intron enhancesexpression of the heterologous polynucleotide in plants.
 11. Arecombinant DNA construct comprising an intron operably linked to apromoter and a heterologous polynucleotide, wherein the intron comprisesa nucleotide sequence that comprises at least one copy of SEQ ID NO: 99,and wherein the intron is capable of enhancing expression of aheterologous polynucleotide in a monocotyledonous plant when compared toa corresponding recombinant DNA construct without the intron.
 12. Therecombinant DNA construct of claim 11, comprising the nucleotidesequence of the intron comprises at least one copy of SEQ ID NO: 100.13. (canceled)
 14. (canceled)
 15. (canceled)
 16. A plant comprising therecombinant DNA construct of claim
 10. 17. A seed comprising therecombinant DNA construct of claim
 10. 18. A plant comprising therecombinant DNA construct of claim
 11. 19. A seed comprising therecombinant DNA construct of claim
 11. 20. A plant comprising therecombinant DNA construct of claim
 12. 21. A seed comprising therecombinant DNA construct of claim
 12. 22. (canceled)
 23. A method foridentifying an intron useful for enhancing transgene expression in amonocotyledenous plant comprising the steps of: (a) scanning a pluralityof monocot introns for the presence of a sequence motif identical to SEQID NO: 99; (b) selecting an intron from step (a) comprising a nucleotidesequence that contains at least one copy of a sequence motif identicalto SEQ ID NO: 99; and (c) measuring the enhancing effect of the intronof step (b) on the expression of an operably linked heterologouspolynucleotide.
 24. (canceled)
 25. A method for modulating transgeneexpression in a plant comprising the steps of: (a) introducing into aregenerable plant cell the recombinant DNA construct of claim 1; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a) wherein the transgenic plant comprises the recombinant DNAconstruct; and (c) obtaining a progeny plant derived from the transgenicplant of step (b), wherein the progeny plant comprises the recombinantDNA construct and exhibits enhanced transgene expression when comparedto a plant comprising in its genome the recombinant DNA constructwithout the corresponding intron sequence.
 26. The method of claim 25wherein said plant is a monocot.